Stranded Total RNA-seq of whole blood cells collected from Holstein Friesian cows that were defined from cluster analysis as being metabolically "balanced", "imbalanced" or "other
Ontology highlight
ABSTRACT: Homeorhetic mechanisms assist dairy cows to transition from pregnancy to lactation. In some cows this is less successful and they develop severe negative energy balance, placing them at risk of metabolic and infectious disease and reduced fertility. We placed Holstein Friesian cows into metabolically BALANCED or IMBALANCED clusters using as biomarkers measurements of plasma nonesterified fatty acids, β-hydroxybutyrate, glucose and IGF-I collected at 14 and 35 day in milk (DIM). To determine underlying mechanisms associated with their metabolic and immune dysfunction, we performed stranded total RNA sequencing using whole blood cells collected from some of the animals.
Project description:Bovine mastitis is an inflammatory disease of the mammary gland with serious economic implications for dairy industries worldwide. We performed total RNA sequencing using whole blood cells collected from multiparous Holstein Friesian dairy cows with naturally occurring mastitis to investigate the changes in systemic gene expression and their association with inflammatory responses. Some related sequencing data are deposited in E-MTAB-9347 and E-MTAB-9348.
Project description:The regulation of endometrial inflammation has important consequences for the resumption of bovine fertility post-partum. All cows experience bacterial influx into the uterus after calving; however a significant proportion fail to clear infection leading to the development of cytological endometritis (CE) and compromised fertility. We hypothesised that early immunological changes could not only act as potential prognostic biomarkers for the subsequent development of disease but also shed light on the pathogenesis of endometritis in the post-partum dairy cow. Here, next-generation sequencing from endometrial biopsies taken at 7 days post-partum (DPP) identified significant expression of inflammatory genes in all cows. Despite the common inflammatory profile and enrichment of the Toll-like receptor, NF?B and TNF signalling pathways, 73 genes and 31 miRNAs differentiated between healthy cows (HC, n=9) and cows which subsequently developed CE at 7 DPP (n=6, FDR<0.1). In healthy cows, 4197 differentially expressed genes between 7 and 21 DPP whereas only 31 genes were differentially expressed in samples from cows with CE. At 21 DPP, a further 1167 genes were differentially expressed between HC cows and cows diagnosed with CE (FDR<0.1). These changes in host gene expression reflected culture-independent microbiological analysis which showed significant differences in uterine bacterial profiles between groups. Inflammatory activity was not confined to the uterus; decreased circulating granulocytes and increased Acute Phase Protein (SAA and HP) plasma expression levels were detected at 7 DPP in cows that developed CE. In conclusion, our data suggests that the major inflammatory cascade activated early post-partum is resolved thereby restoring homeostasis in healthy cows by 21 DPP, but this transition fails to occur in cows which develop CE. Despite a common inflammatory profile, differential expression of specific immune genes may identify cows at risk of prolonged inflammation and the development of CE post-partum. Sixteen Holstein Friesian cows, of mixed parity, within the same university dairy herd were sampled 7 and 21 days postpartum (DPP) in the morning after milking, over an eight week period.
Project description:As part of cross-platform comparisons of microarray and RNA-seq, this current experiment using the Affymetrix Human Transcriptome Array 2.0 aimed to quantify gene-level expression in subjects administered recombinant human erythropoietin over a 10-week protocol for the identification of gene signatures of blood doping. These results were compared to results obtained from other gene expression quantification platforms using the same experimental cohort, including the Illumina HumanHT-12v4 Expression BeadChips (archived in ArrayExpression; E-MTAB-2874), Illumina NextSeq 500 and MGI DNBSEQ-G400RS.
Project description:The regulation of endometrial inflammation has important consequences for the resumption of bovine fertility post-partum. All cows experience bacterial influx into the uterus after calving; however a significant proportion fail to clear infection leading to the development of cytological endometritis (CE) and compromised fertility. We hypothesised that early immunological changes could not only act as potential prognostic biomarkers for the subsequent development of disease but also shed light on the pathogenesis of endometritis in the post-partum dairy cow. Here, next-generation sequencing from endometrial biopsies taken at 7 days post-partum (DPP) identified significant expression of inflammatory genes in all cows. Despite the common inflammatory profile and enrichment of the Toll-like receptor, NFκB and TNF signalling pathways, 73 genes and 31 miRNAs differentiated between healthy cows (HC, n=9) and cows which subsequently developed CE at 7 DPP (n=6, FDR<0.1). In healthy cows, 4197 differentially expressed genes between 7 and 21 DPP whereas only 31 genes were differentially expressed in samples from cows with CE. At 21 DPP, a further 1167 genes were differentially expressed between HC cows and cows diagnosed with CE (FDR<0.1). These changes in host gene expression reflected culture-independent microbiological analysis which showed significant differences in uterine bacterial profiles between groups. Inflammatory activity was not confined to the uterus; decreased circulating granulocytes and increased Acute Phase Protein (SAA and HP) plasma expression levels were detected at 7 DPP in cows that developed CE. In conclusion, our data suggests that the major inflammatory cascade activated early post-partum is resolved thereby restoring homeostasis in healthy cows by 21 DPP, but this transition fails to occur in cows which develop CE. Despite a common inflammatory profile, differential expression of specific immune genes may identify cows at risk of prolonged inflammation and the development of CE post-partum. Sixteen Holstein Friesian cows, of mixed parity, within the same university dairy herd were sampled 7 and 21 days postpartum (DPP) in the morning after milking, over an eight week period.
Project description:the Enhanced Epidermal Antigen Specific Immunotherapy Trial -1 (EEASI) study was a two-centre, open-label, uncontrolled, single-group first-in-human Phase 1A safety study of C19-A3 GNP peptide in individuals with T1D (https://clinicaltrials.gov/ct2/show/NCT02837094). The investigational medicinal product (IMP) was C19-A3 GNP (Midacore™), which comprises Midacore™ GNPs (Midatech Pharma Plc, Cardiff, UK)(1, 20) of a size of less than 5 nm, covalently coupled to an 18-amino acid human peptide, the sequence of which is identical to the residues from position 19 in the C-peptide of proinsulin through to position 3 on the A-chain of the same molecule (GSLQPLALEGSLQKRGIV). The peptide is synthesised with a linker to facilitate binding to the GNPs: 3-mercaptopropionyl-SLQPLALEGSLQKRGIV 2 acetate salt (disulfide bond). The chemical composition of the IMP contained a ratio of 4 C19-A3 peptides: 11 glucose C2: 29 glutathione ligands as determined by 1H-NMR (proton nuclear magnetic resonance). A typical batch contained: [C19-A3 peptide] = 1.33 mg/ml; [gold] = 5.5 mg/ml; [glucose linker] = 0.6 mg/ml; [glutathione linker] = 1.79 mg/ml. As the drug substance was diluted 1:7 to 1:10, depending on the content of C19-A3 peptide per particle, and as 50 μl of the diluted solution was administered to the study participants, this corresponded to C19-A3 peptide: 10 μg; gold: 39 μg; glucose linker: 4.3 μg; glutathione: 12.7 μg. Participants diagnosed with T1D and confirmed to possess the HLA-DRB1∗0401 genotype, were given three doses of C19-A3 GNP at 4-weekly intervals (weeks 0, 4, and 8) in the deltoid region of alternate arms (2 doses in one arm and 1 dose in the other arm) via CE-marked 600 μm length MicronJet600™ hollow microneedles (NanoPass Technologies Ltd. Israel) attached to a standard Luer-lock syringe. The single-dose given in 50 μl volume was equivalent to 10 μg of C19-A3 peptide. Punch skin biopsies of the local area were performed under aseptic conditions and under local anaesthetic (Lidocaine Hydrochloride Injection BP 2%, w/v), using a 6-mm sterile disposable biopsy punch. Following the biopsy, samples were divided with half immediately placed in 10% formalin and transported to the laboratory and the other half placed in RNA laterTM (Fisher Scientific, Loughborough, UK) for bulk RNA sequencing. Control skin samples were obtained from female patients aged 19–82 years, following mastectomy or breast reduction after informed consent. Skin without obvious pathological findings, which was surplus to diagnostic histopathology requirements, was used in the experiments. Control punch biopsy samples were treated in the same manner as described above. After cutting skin samples into small pieces, samples were homogenised and lysed by using Tissue Raptor II and QIAzol® Lysis protocol (QIAzol® Handbook 2021, QIAgen, Crawley, UK). The quality of RNA was routinely assessed by determining the A260:A280 ratio using NanoDrop2000 (Thermo Scientific, UK). RNA libraries were created using TruSeq stranded Total RNA with ribozero GOLD (illumina, UK) and sequenced on an illumina HiSeq 2500 platform with 2 x 75 bp reads
Project description:RNA was extracted from whole blood of subjects collected in Tempus tubes prior to COVID-19 mRNA booster vaccination. D01 and D21 correspond to samples collected at pre-dose 1 and pre-dose 2 respectively. RNA was also extracted from blood collected at indicated time points post-vaccination. DB1, DB2, DB4 and DB7 correspond to booster day 1 (pre-booster), booster day 2, booster day 4 and booster day 7 respectively. The case subject experienced cardiac complication following mRNA booster vaccination. We performed gene expression analysis of case versus controls over time.
Project description:RNA was extracted from whole blood of subjects collected in Tempus tubes on day 0 (immediately prior to vaccination), day 1 and 3 post-vaccination. We performed gene expression analysis of subjects with similar baseline antibody titres to rubella virus (RV) that experienced an increase in anti-RV IgG titre (>= 2-fold) or not (< 2 fold) at one month post-vaccination.
Project description:The cohort comprised patients recruited between Sept 2014- June 2020. It includes patients with bacteremia and positive viral diagnostic test in the context of acute admission. All patients with definite infection were used for signature discovery. Whole blood was collected at the time of recruitment in Tempus Blood RNA tubes and total RNA was isolated with the Tempus Spin RNA Isolation Kit (ThermoFisher Scientific) according to the manufacturer’s instructions. RNA samples were stored at −80 °C until further analysis. After additional DNAse treatment, library preparation and sequencing of 30 million 150bp, paired end reads were conducted using the Illumina's TruSeq® RNA Sample Preparation Kit; ribosomal and globin RNA depletion was performed using the Illumina Ribo-Zero Gold kit and HiSeq 4000 at The Wellcome Centre for Human Genetics in Oxford UK.
Project description:RNA-sequencing was performed on baseline blood samples from HIV-infected and HIV-uninfected asymptomatic adults with recent household exposure to an index case of infectious pulmonary tuberculosis (TB) and with detectable Mtb DNA in PBMC. Additional sequencing was also performed on follow-up blood samples from HIV-infected participants following completion of isoniazid preventative therapy.
Project description:Blood from four healthy volunteers was sampled on both PAXgene and Tempus tubes after two hours of fasting and abstention from coffee, medication and exercise. The content of each tube was split in two aliquotes where one aliquot was isolated using the sampling systems' original protocol and the second aliquot using a modified common protocol. The aim of this study was to establish a common RNA isolation protocol for these two systems and investigate if it could reduce the differences in gene expression.